The overall goal of this application is to clarify the molecular organization and function of enigmatic axonal actin assemblies that my lab recently discovered. Actin organization along axon shafts has been relatively ignored for decades, probably because routine staining ? using the classic actin-filament marker phalloidin ? only shows a patchy, uninteresting pattern. However, recent studies from us and others' using super-resolution microscopy and low-light imaging have revealed a hidden world of actin in axon shafts ? replete with elaborate circumferential rings underneath the plasma membrane, and rapidly elongating linear filaments along the axis of the axon shaft. We have been fortunate to be at the forefront of these discoveries, and our studies have revealed fascinating actin dynamics in axons. Specifically, we found that axons have focal ?hotspots? of actin ? spaced ~ 3-4 m apart ? where actin continuously polymerizes and depolymerizes. These hotspots give rise to long actin filaments that rapidly (and bidirectionally) elongate along the axon shaft (we named these ?actin trails?). Actin rings, trails and hotspots are seen in axons in vivo, and we will use in vivo model systems in this proposal as well. Although the overall assemblies have been seen, molecular events underlying the generation and maintenance of these structures is unclear. Importantly, their functional roles in axons are unknown. Here we propose three specific aims to clarify the organization and function of these newly-discovered actin assemblies: Aim #1: Identify mechanisms initiating actin trails in axons. Aim #2: Identify mechanisms elongating actin trails in axons. Aim #3: Determine functions of axonal actin assemblies.